The Mechanism Of Expression Of B4GALT1 In Bone Marrow Stromal Cells Mediating Drug Resistance Of Malignant Clones

The bone marrow hematopoietic microenvironment is the internal environment that supports and regulates the growth and development of hematopoietic stem/progenitor cells.As one of the important components of bone marrow,stromal cells play an important role in the process of quiescence,differentiation,proliferation and apoptosis of hematopoietic stem cells.Myelodysplastic syndrome(MDS)is a group of myeloid malignant clonal diseases originating from hematopoietic stem cells.Chemotherapy is a routine treatment for high-risk patients with MDS,but clinically,some patients with MDS have different degrees of drug resistance.Studies have shown that in addition to the spontaneous drug-resistance characteristics of malignant clones,abnormal bone marrow hematopoietic microenvironment also contributes to the emergence of drug resistance of malignant clonal cells,but the specific mechanism is still unclear.In the previous work,we found that two bone marrow stromal cells HS5 and HS27 a have significantly different effects on the proliferation of MDS cells through mouse transplantation model.The expression of ?-1,4-galactosyltransferase 1(B4GALT1)in HS27 a was significantly higher than HS5 by glycol-gene chip and protein quantitative mass spectrometry.In this study,we found after co-cultured MDS clonal cells with HS5,the expression of B4GALT1 in co-cultured HS5 was significantly increased.Moreover,MDS cells became more resistant to chemotherapeutic drugs(cytarabine and doxorubicin)after cocultured with HS5 that overexpressing B4GALT1(HS5B4).Thereafter,this study explored the molecular mechanism of differential expression of B4GALT1 in two bone marrow stromal cells,and investigated the molecular mechanism of rendered drug resistance of MDS clonal cells mediated by direct contact with stromal cells that overexpressing B4GALT1,by cytokine secretion and by exosomal secretion from the B4GALT1-expressing stromal cells.The main results are as follows:(1)Molecular mechanisms of differential expression of B4GALT1 in two bone marrow stromal cells.In this study,higher expression of B4GALT1 in HS27 a cells was screened by multi-omics assays.We found B4GALT1 was regulated by JNK/c-Jun/AP-1.We showed higher expression of c-Jun and its phosphorylated form resulted in higher expression of B4GALT1 in HS27 a cells.Further,expression of B4GALT1 and its product LacNAc in the stromal cells from 5 healthy doners and 20 patients with MDS were detected by immunofluorescence staining and immunohistochemistry.We comfirmed the significantly higher expression of B4GALT1 and LacNAc were in stromal cells of patients with MDS than in healthy donors.(2)The molecular mechanism of rendered drug resistance of MDS clonal cells mediated by direct contact with stromal cells that overexpressing B4GALT1.We found overexpression of B4GALT1 in HS5 cells enhanced the adhesion of MDS cells to HS5 cells.MDS cells became more resistant to doxorubicin or cytarabine only after co-cultured with HS5 overexpressing B4GALT1(HS5B4).Further,the more LacNAc modification of DNER in HS5B4 was identified by lectin affinity mass spectrum.DNER is ligand protein of Notch1 which located in cell membrane.With the participation of Galectin-1 in extracellular matrix,LacNAcmodificated DNER can activate Notch1 signaling pathway,and increase the resistance of MDS cells to chemotherapeutic drugs.(3)The molecular mechanism of rendered drug resistance of MDS clonal cells mediated by cytokine secretion from stromal cells overexpressing B4GALT1.In this part,MDS cells treated with conditioned medium derived from HS5B4 showed significantly enhanced resistance to doxorubicin or cytarabine.We found the concentration of chemokine CXCL1 in the conditioned medium derived from HS5B4 was significantly increased by cytokine chip detection.CXCL1 can help MDS cells resisting to chemotheraputic drugs by lowering ROS levels in MDS cell.We showed overexpression of B4GALT1 in HS5 resulted in more LacNAc modification on p53,thereby up-regulated its ubiquitination and its degradation,thus in ture can activate NF-?B signaling pathway and enhance the production of CXCL1.(4)The molecular mechanism of rendered drug resistance of MDS clonal cells mediated by exosomes derived from HS5 cells expressing B4GALT1.In this part,exosomes derived from HS5 and HS5B4 were extracted by ultracentrifugation.MDS cells treated with exosomes derived from HS5B4 were more resistant to doxorubicin or cytarabine than MDS cells treated with exosomes derived from HS5.miRNA microarray showed that expression of miR-92a-3p in exosomes derived from HS5B4 was significantly higher than in exosomes derived from HS5.Therefore,we overexpressed miR-92a-3p in KG1 a and its resistance to chemotherapeutic drugs was increased.